The way cells respond to radiation exposure is important since, if DNA lesions incurred are not properly repaired, deleterious consequences can ensue, including mutagenesis, carcinogenesis or lethality. This application proposes experiments designed to study molecular mechanisms of radioresistance, using the fission yeast Schizoaccharomyces pombe as a model system because this, organism shares many cellular and genetic features with mammalian cells. Specifically, studies will focus on the S. pombe rad9 gene. Mutations within rad9 cause extreme radiosensitivity, indicating that this gene plays a key role in promoting radioresistance. Furthermore, rad9 mutations eliminate the radiation-induced delay in the G2 phase of the cell cycle, normally expressed by wild-type cells and thought to provide extra time for repair before entry into mitosis. Therefore, this gene also serves as a link between radiation exposure and cell cycle regulation. Studies will focus on rad9 alone as well as in the context of the whole cell to learn about its function. It is proposed to systematically mutagenize the gene and test for function in vivo, to localize rad9 regions important for activity. rad9 protein and antibodies will be made to determine the subcellular location of the protein and to identify by immunoprecipitation other proteins that bind specifically to rad9. The two-hybrid system will also be used to identify proteins that bind rad9, and preliminary results indicating that rad9 binds two translation related proteins, EF-1alpha and ribosome protein L9, will be pursued to determine the biochemical and biological consequences of these interactions. In addition, the differential RNA display technique will be used to identify genes whose expression is modulated by rad9. Together, these studies should help define the role of rad9 in radioresistance and cell cycle control and, in a broader sense, the way cells coordinate multiple metabolic pathways to reduce the potentially harmful effects associated with radiation exposure.